The impact of the encapsulation technology on the oxidative stability of fish-oil-loaded capsules was investigated. The capsules (ca. 13 wt% oil load) were produced via monoaxial or coaxial electrospraying and spray-drying using low molecular weight carbohydrates as encapsulating agents . The use of spray-drying technology resulted in larger capsules with higher encapsulation efficiency (EE > 84%), whilst the use of electrospraying produced encapsulates in the sub-micron scale with poorer retention properties (EE < 72%). The coaxially electrosprayed capsules had the lowest EE values (EE = 53–59%), resulting in the lowest oxidative stability, although the lipid oxidation was significantly reduced by increasing the content of pullulan in the shell solution. The emulsion-based encapsulates  presented high oxidative stability during storage, as confirmed by the low concentration of selected volatiles -2,4-heptadienal). Nonetheless, the monoaxially electrosprayed capsules were the most oxidized after production due to the emulsification process and the longer processing time. Food fortification with omega-3 polyunsaturated fatty acids (PUFAs), especially EPA C20:5 n-3) and DHA (C22:6 n-3), has become a focus of scientific research as a result of the recognized health benefits derived from their consumption . However0:5 n-3 aIn oil-loaded capsules, the oil is trapped within a matrix composed of the encapsulating agent/s, which act as a physical barrier against prooxidants  . FurtherSpray-drying is the preferred encapsulation technique in the food industry due to its multiple advantages, such as the high throughput and low cost . FurtherElectrospraying technology is based on applying a high-voltage electrostatic field between the end of an emitter and a grounded collector to cause the ejection of a solution . When thCoaxial electrospraying modifies the monoaxial electrospraying process by introducing a coaxial emitter, which allows to electrospray two liquids simultaneously . In brieLow molecular weight (LMW) carbohydrates such as glucose syrup (GS) and maltodextrin (MD) have been extensively used for the encapsulation of fish oil by spray-drying , and theTaken altogether, the objective of this work was to investigate the production of fish-oil-loaded capsules using different encapsulation technologies such as spray-drying and monoaxial or coaxial electrospraying using GS or MD as the main encapsulating agent. Whey protein concentrate hydrolysate (WPCH), with high emulsifying and antioxidant activity ,16, was ® CITREM LR 10 EXTRA MT) were provided by Danisco . The pullulan (P) was kindly donated by Hayashibara Co., Ltd. . The glucose syrup  was supplied by Cargill Germany GmbH . The maltodextrin  and whey protein concentrate (ca. 35 wt% protein content) were generously donated by Abbott Laboratories S.A. . The whey protein concentrate hydrolysate (WPCH) used as an emulsifier was produced in an automatic titrator  using Alcalase 2.4 L to a degree of hydrolysis of 10% (DH10), as described by Rahmani-Manglano et al. [The refined fish oil (Omega Oil 1812 TG Gold) was purchased from BASF Personal Care and Nutrition GmbH  and stored at −80 °C until use. The Tween-20 (T20) was obtained from Sigma-Aldrich  and CITREM  in the aqueous phase containing the encapsulating agent (GS or MD) and the emulsifier (WPCH or T20). The total solid content of the emulsions was fixed to 34 wt%, leading to a final oil load of the microcapsules of ca. 13 wt%. Therefore, depending on the emulsifier used (WPCH = 6 wt% or T20 = 0.35 wt%), the concentrations of the encapsulating agent varied . The concentration of T20 was optimized to achieve a similar oil droplet size distribution to that of the WPCH-based emulsions (data not shown). The emulsions prepared with WPCH as the emulsifier had a final protein content of 2 wt%, resulting in a protein/oil ratio (P/O ratio) of 0.4. The spray-dried emulsions were produced as follows. First, a coarse emulsion was prepared by adding the oil to the aqueous phase during the first minute of mixing at 15,000 rpm using an Ultraturrax T-25 homogenizer . The total mixing time was 2 min. Then, the pre-emulsion was homogenized in a high-pressure homogenizer  at a pressure range of 450/75 bar and by applying 3 passes. Right after production, the emulsions were subjected to spray-drying in a laboratory-scale spray-drier  at 180 and 90 °C for the inlet and outlet temperatures, respectively. The drying air flow was fixed to 25 NmFirst, fish-oil-in-water emulsions were produced. The aqueous phase consisted of WPCH (4.3 wt%), P (3.0 wt%), and the main encapsulating agent , which were dissolved in distilled water and stirred overnight (500 rpm) at ambient temperature. Then, the CITREM (1 wt%)/fish oil (3.7 wt%) mixture was dispersed in the aqueous phase using a rotor-stator homogenizer prior to high-pressure homogenization, as described in For the coaxial electrospraying, the biopolymer solution flowing through the annular gap between the inner and outer needles was produced following two different approaches. In the first approach, the encapsulating agent , P , and T20 (1 wt%) were dissolved in distilled water and stirred overnight (500 rpm) at ambient temperature before electrospraying. In the second approach, the encapsulating agent , P (3 wt%), and T20 (1 wt%) were dissolved in distilled water and stirred overnight (500 rpm). Afterwards, the biopolymer solution was passed through a high-pressure homogenizer  prior to electrospraying. The electrospraying process was carried out in the setup described in The morphology of the capsules was investigated via scanning electron microscopy (SEM) using an FESEM microscope . Depending on the microencapsulation technology used, a thin layer of microcapsules  or a piece of approximately 0.5 × 0.5 cm aluminum foil containing the sample  was placed on the carbon tape and carbon-coated using an EMITECH K975X Turbo-Pumped Thermal Evaporator . The SEM images were acquired in the range of 500×–15K× magnification with a 5 kV accelerating voltage. The particle size distributions and mean diameters were determined by measuring 180 randomly selected capsules using ImageJ software .2 = 0.99) prepared by dissolving various quantities of fish oil in hexane (0.1–2.0 mg/mL). The EE and was calculated as follows:v/v) solvent. For the extraction, ca. 50 mg of powder was dissolved by adding 10 mL of distilled water, and the total oil load was determined by measuring the absorbance of the lipid extract, as previously described by Rahmani-Manglano et al. [The encapsulation efficiency (EE) of the capsules was measured as described by Prieto and Lagaron  with somo et al. . The meaTo monitor the oxidative stability, 10 mg of capsules was stored in a 2 mL plastic Eppendorf tube at 25 °C in the dark for 6 weeks for the FT-IR analysis. For the measurement of secondary volatile oxidation products, 150 mg of capsules was stored in a 2 mL plastic Eppendorf tube at 25 °C in the dark for 6 weeks. Samples were taken at week 0, 2, 4, and 6 for the analysis.−1 by averaging 100 scans at 2 cm−1 resolution. The pellets were produced in triplicate for each sampling point and the measurements were carried out immediately after pellet production. For the raw ingredients used to produce the capsules , the attenuated total reflection (ATR)–FTIR spectra were recorded with the same equipment and conditions.FT-IR spectra of the capsules were recorded on a JASCO FT/IR 6200  spectrometer operating in transmission mode. Approximately 1.5 mg of capsules was dispersed in ca. 150 mg of spectroscopic-grade potassium bromide (KBr) and subsequently ground. Then, a pellet was formed by compressing the mixture at ca. 150 MPa. All spectra were recorded within the wavenumber range of 4000–400 cmApproximately 50 mg of microcapsules and 5 mg of internal standard  were weighed out in a 100 mL pear-shaped bottle, to which 5 mL of distilled water and 1 mL of antifoam (Synperonic 800 µL/L water) were added. The bottle was heated to 45 °C in a water bath while being purged with nitrogen . Volatile secondary oxidation products were trapped on Tenax GR tubes. The volatiles were desorbed again via heating (200 °C) in an Automatic Thermal Desorber , cryofocused on a cold trap (−30 °C), and released again (220 °C), which led to a gas chromatograph . The oven program had an initial temperature of 45 °C for 5 min, increasing by 1.5 °C/min until 55 °C, 2.5 °C/min until 90 °C, and 12.0 °C/min until 220 °C, where the temperature was kept for 4 min. The individual compounds were analyzed using mass-spectrometry . The individual compounds were identified via both 186 MS library searches  and using authentic external standards, which was quantified through calibration curves. The external standards employed were 2-ethylfuran, 1-penten-3-ol, hexanal, heptanal, and -2,4-heptadienal , and the standard solutions were directly injected into the Tenax tubes. The samples were analyzed in triplicate.p < 0.05) to determine significant differences between mean values.The data were subjected to a one-way analysis of variance (ANOVA) using Statgraphics version 5.1 . Tukey’s HSD multiple range test was used at the 95% confidence level (p > 0.05), and more than 90% of the particles had a size below 20 µm  A,B did nInterestingly, despite the differences in the formulation of the biopolymer-based shell solutions (pullulan content) and the production process , the morphologies of the coaxially electrosprayed capsules were fairly similar among the samples C–F. For p < 0.05) and throughput of the process (from ca. 80 mg/h for -co capsules to ca. 143 mg/h for -HPH-co capsules). Nonetheless, although the infusion flow rate increased by up to two times for the production of -HPH-co systems compared to -co systems, the size of the capsules did not increase in the same proportion as a consequence of the higher voltage applied during the -HPH-co capsule production process (ca. 18 kV for -HPH-co over ca. 13.5 kV for -co electrospraying). Higher processing voltages favor jet breakage into smaller droplets due to increased electrostatic charge repulsion, leading to smaller capsules after solvent evaporation [Overall, regarding the electrosprayed systems , it should be noted that coaxially electrosprayed capsules showed more and larger dents on the capsules’ surface compared to monoaxially electrosprayed systems . The morp < 0.05) were observed in the EE values depending on the encapsulation technique used to produce the fish-oil-loaded capsules  (Significant differences (praying) . The higFor emulsion-based encapsulation techniques, such as spray-drying and monoaxial electrospraying, the EE is closely related to the infeed emulsion formulation and its physical stability. The drying of physically stable monodisperse emulsions of small oil droplets results in the better entrapment of the oil within the encapsulating matrix ,23. Furtp < 0.05) as a result of the different atomization processes of both techniques could further explain the lower EE values reported for the -mo systems over the spray-dried capsules (p < 0.05).For monoaxial electrospraying, the emulsions were produced by dispersing the oil phase, consisting of a CITREM (1 wt%)/fish oil (3.7 wt%) mixture, within the aqueous phase, containing the encapsulating agent , the emulsifier , and pullulan (3 wt%). Both the presence of pullulan in the aqueous phase and CITREM in the oil phase may have increased their respective viscosities to an extent, resulting in poorer emulsification, leading to emulsions of larger oil droplets. Moreover, although the electrosprayed emulsions were gently stirred during the total processing time to minimize their physical destabilization, this was not totally achieved see . After 3Nonetheless, the EE values reported for the capsules produced by monoaxial electrospraying in the current work contrast with previous studies on the encapsulation of fish oil by monoaxial electrospraying within carbohydrate-based matrices. García-Moreno et al. ,11 produp > 0.05) , regardless of the shell solution formulation or production process (-co or -HPH-co capsules) ( > 0.05) . These l > 0.05) C–F, whicOverall, our EE results for the electrospraying processes  contrast with those reported by other authors ,26. PéreFirst, the oxidative stability of the different types of capsules was evaluated via FT-IR by monitoring the changes in the characteristic absorption bands of omega-3 PUFAs during the storage time. Although information on the initial oxidation status of the encapsulated oil is not provided by FT-IR analysis, this technique has been proven to be a useful tool to evaluate the oxidation stage of neat edible oils ,29 and h−1, and a decrease in the intensity of this band (due to the loss of cis double bonds) has been proposed as a marker of lipid oxidation [−1, assigned to rocking vibrations of C–H bonds of cis-disubstituted alkenes, was used as the internal standard for normalization purposes, since this band remains unchanged during the lipid oxidation of omega-3 PUFA-rich oils , as reported by other authors [3012/A1456) and the results were normalized to the initial relative absorbance value (week 0) for a better comparison among the samples. The ATR-FTIR spectra of the fish oil, the GS, and the T20 used in the study are shown in −1 does not overlap with the infrared bands of the other ingredients. The spectra of the rest of the ingredients used in the production of the capsules also did not overlap with the fish oil band at 3012 cm−1, as can be seen elsewhere [For omega-3 PUFA-rich oils, the characteristic absorption band corresponds to the stretching of cis-alkene groups (–HC=CH–) at 3012 cmxidation . Thus, tlsewhere ,12.−1). Likewise, García-Moreno et al. [−1 in fish-oil-loaded capsules produced via monoaxial electrospraying using dextran or GS as the encapsulating biopolymer after 21 days of storage at 20 °C. Nonetheless, p > 0.05)  . TherefoTo further investigate the oxidative stability of the fish-oil-loaded capsules, the contents of selected secondary volatile oxidation products (SVOPs) were measured during 6 weeks of storage at 25 °C. . Volatilp > 0.05). Furthermore, irrespective of the SVOP considered -2,4-heptadienal), the same oxidation trend could be observed for the -co capsules. First, a slight increase in the concentration occurred during the first two weeks of storage, followed by a sharp increase from week 2 onwards (p > 0.05).Considering the results obtained for the SVOPs derived from the oxidation of omega-3 PUFAs A–C, the  onwards A–C. Nonep < 0.05), result in an increased contact area with environmental prooxidants , which facilitates their diffusion through the encapsulating matrix, thereby promoting lipid oxidation [Oxygen’s solubility and diffusion through the encapsulating wall have been proven to play a key role in the lipid oxidation of encapsulated fish oil . Both arxidation . Additioxidation . Moreovexidation . Therefop < 0.05). As previously mentioned, the monoaxially electrosprayed emulsions contained CITREM, which according to the supplier, was made from refined sunflower oil. Sunflower oil is rich in omega-6 PUFAs, and both hexanal and heptanal have been identified in neat sunflower oil [p < 0.05). In addition, the monoaxially electrosprayed emulsions were stored between batches for a total processing time of 36 h, which resulted in physical destabilization, and consequently in a relatively high content of non-encapsulated surface oil (p < 0.05). This could be explained on the basis of aldehyde decomposition as a result of the so called non-enzymatic browning reactions taking place in the presence of the WPCH amino groups [Interestingly, the monoaxially electrosprayed capsules were the most oxidized after processing, as shown by their significantly higher content in the selected volatiles studied at the beginning of the storage time  or maltodextrin (MD)) as the encapsulating agents. Furthermore, in the study, fish-oil-loaded capsules were also produced by spray-drying and monoaxial electrospraying in the presence of GS or MD, using whey protein concentrate hydrolysate (WPCH) as the emulsifier. Our results show that the physicochemical properties of the capsules  were significantly influenced by the encapsulation technology. As expected, larger capsules were produced using spray-drying over electrospraying technology, and the particle size distribution was also broader. Moreover, the use of emulsion-based encapsulation methods  resulted in better entrapment of the fish oil within the encapsulating matrix (EE > 69%) compared to coaxial electrospraying (EE = 53–59%), due to the lack of surface-active properties of the main encapsulating agents used . This explained the lowest oxidative stability observed for the coaxially electrosprayed capsules. However, despite the higher content of non-encapsulated oil in the aforementioned systems, their oxidative stability could be significantly enhanced by increasing the content of pullulan in the shell solution. This was attributed to the larger size of the resulting capsules and to the higher content of pullulan in the encapsulating matrix, both influencing the oxygen diffusivity through the encapsulating wall. The use of emulsion-based encapsulation methods led to capsules with high oxidative stability, as confirmed by the low contents of SVOPs -2,4-heptadienal) found at the end of the storage time. Nonetheless, the monoaxially electrosprayed capsules were the most oxidized right after production due to the emulsification process, together with a larger time span between the emulsion production and drying.


Kidney transplant recipients have an increased risk of complications from COVID-19. However, data on the risk of allograft damage or death in kidney transplant recipients recovering from COVID-19 is limited. In addition, the first and second waves of the pandemic occurred at different times all over the world. In Turkey, the Health Minister confirmed the first case in March 2020; after that, the first wave occurred between March and August 2020; afterward, the second wave began in September 2020. This study aims to demonstrate the clinical presentations of kidney transplant recipients in the first two waves of the pandemic in Turkey and explore the impact of COVID-19 on clinical outcomes after the initial episode.Patients with COVID-19 from seven centers were included in this retrospective cohort study. Initially, four hundred and eighty-eight kidney transplant recipients diagnosed with COVID-19 between 1 March 2020 to 28 February 2021 were enrolled. The endpoints were the occurrence of all-cause mortality, acute kidney injury, cytokine storm, and acute respiratory distress syndrome. In addition, longer-term outcomes such as mortality, need for dialysis, and allograft function of the surviving patients was analyzed.p < 0.001), acute kidney injury , acute respiratory distress syndrome , and cytokine storm rate  were higher in first wave compared to the second wave. These 47 patients died within the first month of COVID-19. Six (1.4%) of the surviving patients lost allografts during treatment. There was no difference in the median serum creatinine clearance of the surviving patients at baseline , first- , third-  and sixth-months . Development of cytokine storm and posttransplant diabetes mellitus were independent predictors for mortality.Four hundred seventy-five patients were followed up for a median of 132 days after COVID-19. Forty-seven patients (9.9%) died after a median length of hospitalization of 15 days. Although the mortality rate (10.1% vs. 9.8%) and intensive care unit admission (14.5% vs. 14.5%) were similar in the first two waves, hospitalization (68.8% vs. 29.7%; Mortality remains a problem in COVID-19. All the deaths occur in the first month of COVID-19. Also, acute kidney injury is common in hospitalized patients, and some of the patients suffer from graft loss after the initial episode. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that caused coronavirus disease 2019 (COVID‐19) was detected in China in December 2019, then triggered the pandemic in March 2020 . LymphopKidney transplant recipients have a high risk of complications from COVID-19 because of immunosuppression and comorbid diseases , 7. Howen = 7) or still under treatment due to COVID-19 (n = 6). Four hundred and seventy-five participants were followed up for at least 28 days or until death, as shown in Fig. This retrospective cohort study included kidney transplant recipients with COVID-19 from seven centers in Turkey. Initially, four hundred and eighty-eight kidney transplant recipients diagnosed with COVID-19 between 1 March 2020 to 28 February 2021 were enrolled. The exclusion criteria included patients who were lost to follow-up after diagnosis , clinical characteristics , and medication during COVID-19  were extracted from electronic medical records. Data from out and hospitalized patients were collected. Patients admitted to the hospital excluded those requiring intensive care unit admission.The patients were diagnosed with COVID-19 based on clinical and laboratory findings. Positive results on real-time polymerase chain reaction (RT-PCR) assay of nasopharyngeal swab specimens or compatible findings on a computed chest tomography scan were used to confirm the diagnosis. In the first three months of the pandemic, patients with a high clinical suspicion of disease were diagnosed by computed tomography of the chest due to low RT-PCR sensitivity ), fever (n = 249 [52.4%]), and dyspnea (n = 162 [34.1%]) were the frequent symptoms at admission, shown in Table n = 92 [21.5%] vs. n = 3 [6.4%]; p < 0.006) was more common in the survivor group than in the non-survivor group. The oxygen saturation at presentation was lower in deceased patients compared to the surviving patients . Lymphocyte count was lower in the deceased patients compared to the survivor patients . Although all serum acute phase reactants were higher in deceased patients compared to the surviving patients (Table p = 0.001), serum ferritin levels , serum interleukin-6 levels , and serum procalcitonin levels . The median serum creatinine levels at admission were similar between the survivor and non-survivor groups .Cough (p = 0.005). Steroid , anakinra , convalescent plasma  were used for the treatment of cytokine storm. While 344 (72.4%) of the patients received anticoagulant treatment, 248 (52.2%) received antibiotics. Duration of hospitalization was longer in deceased patients compared to the survivor patients .Antimetabolites were discontinued in 428 (90.1%), and both antimetabolites and calcineurin inhibitors were stopped in 49 (10.3%) of the patients. Also, favipiravir was used in 45 (95.7%) non-survivors and 346 (80.8%) survivor patients (Forty-seven patients (9.9%) of the participants died after a median hospitalization of 15 days . Forty-six of the forty-seven patients (97.9%) who were admitted to the ICU, and received mechanical ventilation, are shown in Table n = 73 [17.1%] vs. n = 14 [29.8%]; p = 0.032). However, stage two  and stage three acute kidney injury  were more common in deceased patients than in the survivors. Eight patients (1.9%) in the survivor group and eleven patients (23.4%) in the non-survivor group received dialysis treatment during the COVID-19 follow-up (p < 0.001). The occurrence of cytokine storm  and acute respiratory distress syndrome  was higher in the non-survivor group compared to the survivor group.One hundred and sixty-eight (35.3%) patients developed acute kidney injury. There was no difference between the groups in terms of the development of stage one acute kidney injury , acute kidney injury , acute respiratory distress syndrome , and cytokine storm rate  were higher in the first wave compared to the second wave shown in Table The mortality rates were 10.1% and 9.8% in the first and second waves, respectively Fig.  and 5. H  days from the diagnosis of COVID-19. No patient died after the first month of COVID-19 diagnosis. Six (1.4%) of the surviving patients lost allografts during treatment for COVID-19. All were in the pre-dialysis period (eGFR 15–30 mL/min), and immunosuppression except for steroids was discontinued due to severe COVID-19. Acute rejection was diagnosed in two patients (0.5%) after COVID-19. The first patient was examined for graft dysfunction (eGFR: 16 mL/min) that persisted for forty days after COVID-19, and de novo DSA was detected. Glomerulitis and peritubular capillaritis were detected in the allograft biopsy. Allograft function returned to baseline (eGFR:65 mL/min) with plasmapheresis and IVIG treatment. In the second patient, interstitial inflammation and tubulitis were detected in the kidney biopsy performed due to graft dysfunction (eGFR: 24 mL/min) that persisted for twenty days after COVID-19, and 500 mg methylprednisolone treatment was administered for three days. The graft function improved (eGFR:74 mL/min) and returned to baseline.One hundred thirty-eight and three hundred sixty-nine patients were followed for more than six and three months, respectively.There was no difference in the median serum creatinine clearance of the surviving patients at baseline , first- , third-  and sixth-months .P = 0.004]). Multivariate logistic regression analysis revealed that the development of cytokine storm  was an independent predictor for mortality, shown in Table Patients with posttransplant diabetes mellitus were associated with a twofold increased risk of death , where patient care is standardized and improved, but a significant difference is not observed in cases where the hospitals are overcrowding, and therefore patient care cannot be improved, such as in India –29. The The new antiviral agents are still under investigation in COVID-19. Remdesivir, which is currently widely used in most countries, has reduced the length of stay in hospitalized patients . Also, fOur study has several limitations; the follow-up period was short, it did not include the control group or pediatric cases, and PCR samples were collected from symptomatic patients rather than all patients. Also, our findings were obtained from unvaccinated kidney transplant recipients; therefore, our results may differ from infections with new SARS-CoV-2 variants and unvaccinated or partially vaccinated patients. In addition, due to the high false-negative results of RT-PCR in the first three months of the pandemic, some patients were diagnosed with COVID-19 by computed tomography alone. Also, data on the tracking of SARS-CoV-2 variants, antibody measurements, and long-term symptoms of COVID were not collected. These limitations suggested that we could not draw firm conclusions from these experiences; therefore, our findings should be validated by prospective cohort studies. Using a standard immunosuppressive treatment regimen can be a strength of the study.In conclusion, this study shows that cytokine storm was a risk factor for death. The deaths occurred in the first month of COVID-19. Also, acute kidney injury is common in kidney transplant recipients with COVID-19, and some of them suffer from allograft rejection and graft loss after the initial acute episode.


In critically ill patients, positive fluid balance is associated with excessive mortality. The POINCARE-2 trial aimed to assess the effectiveness of a fluid balance control strategy on mortality in critically ill patients.POINCARE-2 was a stepped wedge cluster open-label randomized controlled trial. We recruited critically ill patients in twelve volunteering intensive care units from nine French hospitals. Eligible patients were ≥ 18 years old, under mechanical ventilation, admitted to one of the 12 recruiting units for > 48 and ≤ 72 h, and had an expected length of stay after inclusion > 24 h. Recruitment started on May 2016 and ended on May 2019. Of 10,272 patients screened, 1361 met the inclusion criteria and 1353 completed follow-up. The POINCARE-2 strategy consisted of a daily weight-driven restriction of fluid intake, diuretics administration, and ultrafiltration in case of renal replacement therapy between Day 2 and Day 14 after admission. The primary outcome was 60-day all-cause mortality. We considered intention-to-treat analyses in cluster-randomized analyses (CRA) and in randomized before-and-after analyses (RBAA).p = 0.26]. Among safety outcomes, only hypernatremia was more frequent in the strategy group . The RBAA led to similar results.A total of 433 (643) patients in the strategy group and 472 (718) in the control group were included in the CRA (RBAA). In the CRA, mean (SD) age was 63.7 (14.1) versus 65.7 (14.3) years, and mean (SD) weight at admission was 78.5 (20.0) versus 79.4 (23.5) kg. A total of 129 (160) patients died in the strategy (control) group. Sixty-day mortality did not differ between groups [30.5%, 95% confidence interval (CI) 26.2–34.8 vs. 33.9%, 95% CI 29.6–38.2, The POINCARE-2 conservative strategy did not reduce mortality in critically ill patients. However, due to open-label and stepped wedge design, intention-to-treat analyses might not reflect actual exposure to this strategy, and further analyses might be required before completely discarding it.Trial registration POINCARE-2 trial was registered at ClinicalTrials.gov (NCT02765009). Registered 29 April 2016.The online version contains supplementary material available at 10.1186/s13054-023-04357-1. Causes of positive fluid balance, observed in most critically ill patients , includeThe effectiveness of interventions controlling fluid balance on survival remains unclear. Two randomized controlled trials of patients with ARDS showed that a strategy of fluid balance control  increased the number of mechanical ventilator-free days (MVFDs), and reduced ICU length of stay with no noticeable adverse effects , 16. AdjComité de Protection des Personnes Est III, Grand-Est, North-East France, has reviewed and approved POINCARE-2 trial (ID-RCB: 2015-A00662-47).The protocol of POINCARE-2 (POids INtensive CARE 2) trial has been extensively detailed elsewhere . BrieflyBecause of (i) the complex design of the intervention , and (ii) the open-label design; and to address the potentially resulting contamination bias , we opted for a cluster-randomized trial over an individual randomized trial. In addition, due to the amount of evidence suggesting the deleterious effect of fluid imbalance on survival and to the relatively easy-to-implement aspect of the strategy in daily ICU practice, we opted for a stepped wedge design to allow each participating ICU to eventually implement the strategy under scrutiny. A stepped wedge trial is a type of cluster-randomized trial where clusters (ICUs) recruit patients both in the control group during the first period (semester) of recruitment and in the strategy group during the last period (semester) of recruitment; and where the switch between control and strategy recruitment is set at a randomly assigned time (wedge).For feasibility purposes, the recruitment period was restricted to 1 year in each ICU. Relying on a previously published design , we handWe presented the trial design in Fig. Eligible ICUs were French ICUs that volunteered to participate in the trial.Inclusion criteria for patients were: age ≥ 18 years old, under mechanical ventilation , admission to one of the 12 recruiting ICUs for > 48 and ≤ 72 h, and expected length of stay after inclusion > 24 h. Exclusion criteria were clinical condition or unavailability of bedside scale impeding weighing, multiple trauma, hospital stay in another ICU > 24 h immediately preceding the index ICU admission, pregnancy, expected withdrawal of life-sustaining therapy within 7 days after admission, patient refusal of personal data collection (or use), history of ICU stay in one of the 12 recruiting ICUs during the recruitment period, and under guardianship.One of the investigators of the concerned ICU informed eligible patients and their kin about the trial protocol and their right to refuse to participate in the trial. Because the POINCARE-2 strategy focused on health care organization, written informed consent was waived in accordance with the French law .Assignment of the POINCARE-2 strategy was at the cluster level and performed by JMV. PEB recruited volunteer ICUs. JMV used computer-generated numbers to randomly assign each ICU to one of the 12 sequences. ICU staff were informed about their own allocation 1 month before their time of inclusion. ICUs were blinded to other ICUs allocation . Once an ICU was assigned to a sequence, the research team met with the whole ICU staff and the dedicated onsite research nurse to inform them about the trial procedures for the control period during the month before the time of inclusion. The control period started at this time at a cluster level. The control period was followed by a training period . All participants recruited over one cluster-period were included in either the control or the strategy group, depending on date of admission to the recruiting ICU Fig. .The POINCARE-2 strategy is summarized in Additional file Patients of the control group received standard care, as routinely delivered by each ICU staff in usual practice.2:FiO2 (mmHg) were collected daily, and their worst value was considered in case of multiple assessment per day. Treatment with vasopressors, renal replacement therapy and mechanical ventilation were also collected daily until Day 60.Baseline patient characteristics collected consisted of age, sex, weight (kg), Mc Cabe score , coexisting conditions, Simplified Acute Physiology Score (SAPS) II, Sepsis-related Organ Failure Assessment (SOFA), and main cause of admission to the ICU. Serum sodium level (mmol/L), serum potassium level (mmol/L), serum bicarbonate level (mmol/L), serum creatinine level (mg/dL), and arterial PaOIndependent research nurses collected daily body weight, biological assays, and treatment prescriptions from Day 0 to Day 14 from medical records. To assess contamination during the control period , data concerning components of POINCARE-2 strategy  were also collected by independent research nurses for patients included during the control period. Weight was routinely collected daily from Day 0 to Day 14 in the strategy group, and on Day 0, Day 7, and Day 14 in the control group (or more frequently when weighing was performed more frequently as part of routine care during the control period).To assess adherence to the POINCARE-2 strategy once the strategy was implemented, we used fluid balance estimates and furosemide and albumin prescription as indicators of adherence. Fluid balance was assessed by using (i) weight evolution between Day 0 and Day 7, defined as the difference between weight at Day 7 and weight at Day 0; (ii) weight evolution between Day 0 and Day 14, defined as the difference between weight at Day 14 and weight at Day 0; (iii) average fluid balance from Day 0 to Day 7, defined as individual arithmetic mean of daily fluid balance  over 7 days after Day 0; and (iv) average fluid balance from Day 0 to Day 14, defined as individual arithmetic mean of daily fluid balance  over 14 days after Day 0. Furosemide and albumin prescriptions were assessed using (i) total dose of diuretics from Day 0 to Day 14, defined as the cumulated dose of diuretics administered to a patient over 14 days after Day 0, and (ii) total dose of albumin 20% from Day 0 to Day 14, defined as the cumulated dose of albumin 20% administered to a patient over 14 days after Day 0.The primary outcome was 60-day all-cause mortality. Vital status and date of event in case of death were extracted from medical records or collected by investigators by phone calls to patients or their family.Secondary outcomes were 28-day and in-hospital all-cause mortality; MVFDs, defined as the cumulative number of days alive with no mechanical ventilation between Day 0 and Day 28; vasopressor-free days (VFDs), defined as the cumulative number of days alive with no prescription between Day 0 and Day 28; and renal replacement therapy-free days (RRTFDs), defined as the cumulative number of days alive with no renal replacement therapy between Day 0 and Day 60. Patients dying within the assessment intervals, and who were under mechanical ventilation, vasopressor prescription, or renal replacement therapy at the time of death, were assigned a count of zero free days, respectively, for the variable of interest.Safety outcomes consisted of unexpected harmful events occurring at least once between Day 2 and Day 14: arterial hypotension, defined as arterial systolic pressure < 90 mmHg; hypernatremia, defined as serum sodium level > 155 mmol/L; hypokalemia, defined as serum potassium level < 2.8 mmol/L; renal damage, defined by a worsening in the RIFLE criteria during Day 3–Day 14 compared to the higher RIFLE criteria during Day 1 and Day 2 ; and acuWe split analyses between CRA  and RBAA . All analyses were intention-to-treat.Baseline characteristics and adherence to the strategy were described in each group (strategy vs. control), and compared by using the absolute value of the standardized difference (Stdiff), with a Stdiff > 0.1 considered as a marker of imbalance between groups. We described categorical variables with frequencies and percentages, and continuous variables with mean and standard deviation (SD) in case of normal distribution, or median and interquartile range (IQR) otherwise.We described outcomes in each group (strategy vs. control) with frequencies and percentages for categorical variables, median and IQR for continuous variables, or Kaplan Meier estimates for survival.To assess the effect of the strategy, we used log-binomial mixed models (or modified Poisson regressions in case of non-convergence) for binary outcomes, and zero-inflated negative binomial (or zero-inflated Poisson) mixed models for continuous outcomes. We relied on two-level crude models, with ICU (and 2-month period effect in RBAA only) entered as a random effect and intervention group as a fixed effect. As planned, we entered additional unbalanced baseline characteristics  as fixed effects in adjusted models. We presented the results as exp(parameters) with their 95% confidence intervals (CIs). For binary outcomes analyzed with log-binomial or modified Poisson mixed models, exp(parameters) can be interpreted as risk-ratios [RRs]. For continuous outcomes analyzed with zero-inflated models, exp(parameters) can be interpreted as odds ratios (OR) for the logistic zero-excess part of the model and as a multiplicative factor of the counting process for the negative binomial part of the model.p-values were two-sided, and statistical significance was set at 0.05 for the primary outcome in the main analyses, and for safety outcomes. In all other analyses, we used Bonferroni adjustment. We performed all analyses using SAS© 9.4 .We imputed missing outcomes based on the maximal bias hypothesis. In multivariable analyses, we treated missing unbalanced baseline characteristics by multiple imputation. k of 0.26, alpha set at 0.05, a power set at 0.8, and a minimal 15% absolute reduction [Assuming an expected 60-day mortality of 37.5% –24 in theduction  in mortaWe monitored data for all included patients while the trial was ongoing (list of queries available upon request).The POINCARE-2 trial was registered at ClinicalTrials.gov .A total of 10,272 patients were admitted to one of the 12 recruiting ICUs over the recruitment period held from June 1, 2016, to May 31, 2019   was administered to 314 patients in the strategy group versus 305 mg (IQR 80–727) to 328 patients in the control group. Over the same period, a median of total infused 20% albumin of 550 mL (IQR 300–1000) was administered to 60 patients in the strategy group versus 400 mL (IQR 300–1000) to 47 patients in the control group. Similar results were observed in RBAA, except for furosemide, which was no longer less prescribed in the strategy than control group [233 mg (IQR 80–590) to 478 patients vs. 220 mg (IQR 80–620) to 475 patients, respectively, Additional file Results for the primary, secondary, and safety outcomes of CRA (RBAA) are summarized in Table p = 0.26, Table p = 0.99, Table A total of 129 patients died in the strategy group (vs. 160 in the control group) during the first 60 days of follow-up. Three patients were lost to follow-up in the strategy group and further considered dead . Survival estimates in the 2 groups are displayed in Additional file In CRA, 28-day and in-hospital mortality, MVFDs, VFDs, and RRTFDs did not differ between groups (Table p = 0.01). Adjustment for potential confounders did not modify this result . In addition, CRA results showed that furosemide was prescribed more in the control than strategy group, which could result from a greater need of diuretics in patients without water/salt restriction, or by residual unbalance between groups. Because furosemide was slightly (though not significantly) more prescribed in the strategy group in RBAA  weight difference and average fluid balance were positive but lower than expected, as compared to that observed in trials included in the meta-analysis by Silversides et al . Five ofAlthough the timing for fluid management was strict in the POINCARE-2 strategy, the strategy itself was more of a set of recommendations than a restrictive standardized procedure. Although hypothetical, intensivists’ attitudes toward the POINCARE-2 strategy could explain part of the observed contamination: in other words, they might have been convinced of the beneficial effect of a conservative approach before the POINCARE-2 trial started, and might have implemented it earlier than planned during the trial. Only a complete process evaluation  could hePotential deleterious consequences of conservative fluid management or de-resuscitation include hemodynamic disturbances, deterioration of renal function, and electrolyte disturbances. The occurrence of arterial hypotension, major ischemic events, and renal damage, as well as VFDs and RRTFDs, did not differ between groups, in both CRA and RBAA. Altogether, these results suggest that a conservative approach was safe for hemodynamic and renal function.Severe hypernatremia was more frequent in the strategy group whatever the analyses. Similar results were observed in the trial conducted by the ARDS network , and a rCIC, Epidémiologie clinique) ensured their accuracy. Third, additional process evaluation, as-treated, and subgroup analyses were planned a priori, and will help further discuss the implementation, underpinning mechanisms of the strategy, context of implementation, and actual effectiveness of the POINCARE-2 strategy with regard to the results presented in this article.The POINCARE-2 trial has some strengths. First, the number of critically ill patients were prospectively included in a timely manner, as planned, which is critical in a stepped wedge trial and could otherwise compromise the design. Second, the thorough monitoring of the data by an ISO 9001-certified research lab . Table S1. Baseline characteristics of patients in randomized before-and-after analyses of the POINCARE-2 trial. Table S2. Description of missing data at baseline for patients in cluster-randomized (CRA) and randomized before-and-after (RBAA) analyses of the POINCARE-2 trial. Table S3. Adherence to the POINCARE-2 strategy in cluster-randomized and randomized before-and-after analyses. Fig. S2. Evolution of the average weight from Day 0 to Day 14 in patients in randomized before-and-after analyses of the POINCARE-2 trial . Fig. S3. Smoothed cumulated fluid balance evolution from Day 0 to Day 14 in patients in cluster-randomized analyses of the POINCARE-2 trial . Table S4. Primary, Secondary, and Safety Outcomes in randomized before-and-after analyses of the POINCARE-2 trial. Table S5. Intra-cluster, intra-period and intra-cluster-period correlation for 60-day mortality. Fig. S4. Smoothed cumulated fluid balance evolution from Day 0 to Day 14 in patients in randomized before-and-after analyses of the POINCARE-2 trial . Fig. S5. Survival of patients included in cluster-randomized analyses of the POINCARE-2 trial. Fig. S6. Survival of patients included in randomized before-and-after analyses of the POINCARE-2 trial.


Paris polyphylla Sm. (C-P) drug pair in the treatment of adenomyosis (AM) based on network pharmacology and animal experiments.  To explore the molecular mechanism of the Cinnamomi ramulus and  Via a network pharmacology strategy, a drug-component-target-disease network (D-C-T-D) and protein–protein interaction (PPI) network were constructed to explore the core components and key targets of C-P drug pair therapy for AM, and the core components and key targets were verified by molecular docking. Based on the results of network pharmacology, animal experiments were performed for further verification. The therapeutic effect of the C-P drug pair on uterine ectopic lesions was evaluated in a constructed AM rat model.  A total of 30 components and 45 corresponding targets of C-P in the treatment of AM were obtained through network pharmacology. In the D-C-T-D network and PPI network, 5 core components and 10 key targets were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the PI3K signaling pathway was the most significantly enriched nontumor pathway. Molecular docking showed that most of the core components and key targets docked completely. Animal experiments showed that the C-P drug pair significantly ameliorated the pathological changes of endometriotic lesions in AM model rats and inhibited PI3K and Akt gene expression, and PI3K and Akt protein phosphorylation. In addition, treatment with the C-P drug pair promoted AM cell apoptosis; upregulated the protein expression of Bax, Caspase-3, and cleaved Caspase-9; and restrained Bcl-2 expression.  We propose that the pharmacological mechanism of the C-P drug pair in the treatment of AM is related to inhibition of the PI3K/Akt pathway and promotion of apoptosis in AM ectopic lesions. Adenomyosis (AM) is a gynecological disorder characterized by the abnormal appearance of endometrial glands and stroma in the myometrium. Increased dysmenorrhea, abnormal uterine bleeding (AUB), and infertility are the most typical symptoms of AM and severely affect the quality of life of patients . HormoneParis polyphylla Sm. (Chonglou) is recorded in Shennong's Herbal Classic (Shen Nong Ben Cao Jing) and is believed to be able to relieve heat, remove toxic material, detumescence, and analgesic effects. Gongxuening capsules , which are commonly used in the clinical treatment of AM, are developed from Paris polyphylla Sm. and have been used to treat uterine diseases and blood stasis syndrome for decades [Among alternative medicines, traditional Chinese medicine (TCM) has accumulated considerable clinical experience in AM treatment –7. Cinna decades –12.Paris polyphylla Sm. (C-P), from Guizhi-Fuling Wan and Gongxuening capsules as a drug pair and explored their synergistic regulatory effects on an AM rat model in this study [Paris polyphylla Sm. and Panax notoginseng (Sanqi) reduced the levels of matrix metalloproteinases (MMP-2 and MMP-9), increased the expression of Bax and Caspase-3, and significantly reduced the proliferation and invasion ability of endometrial cells in AM model rats [In TCM treatment theory, prescriptions based on the concept of “drug pairs” can often achieve better results than individual drug prescriptions, as two drugs can often increase the effectiveness of each other. We extracted the monarch drugs (the drugs that play major roles in a formula), namely, Cinnamomi ramulus and is study . A previdel rats . Using CTCMs are characterized by multiple components and multiple targets, which makes it difficult to further study the mechanism of the C-P drug pair in the treatment of AM. In recent years, the emergence of network pharmacology has provided an effective means to study the pharmacological mechanisms of TCMs. Construction and analysis of drug–component–target–disease (D-C-T-D) networks can enable the core components of TCMs and potential targets for disease treatment to be effectively discovered . In thishttps://old.tcmsp-e.com/index.php). The active ingredients obtained from the TCMSP database were screened under the conditions of oral bioavailability (OB) ≥30% and druglikeness (DL) ≥0.18, and the corresponding targets of the active ingredients were further obtained. The ingredients and corresponding targets of Paris polyphylla Sm. were obtained from the HERB database (https://herb.ac.cn/) and a literature search [https://www.swisstargetprediction.ch/) was used to predict the targets for the components obtained from the literature search [The ingredients and targets of Cinnamomi ramulus were obtained from the TCMSP database , Coremine , and PharmGKB (https://www.pharmgkb.org/) databases using “Adenomyosis” as the retrieval keyword. The correlation score was used as a reference for each included target. The common targets between C-P and AM were identified using a Venn diagram.AM-related targets were searched in the GeneCards , and “Homo sapiens” was used as the screening criterion to obtain the PPI relationship [The overlapping targets between the C-P active ingredient targets and the AM-related targets were imported into the STRING database (tionship . The reshttps://david.ncifcrf.gov/) was utilized to perform GO biological process enrichment and KEGG pathway analysis on the overlapping targets between C-P and AM [DAVID 6.8 (P and AM . The tophttps://pubchem.ncbi.nlm.nih.gov/) database. The 3D structures of key target proteins were downloaded from the RCSB database (https://www.rcsb.org/search). Chem3D software was used to preprocess the components, and AutoDock Vina software was used for molecular docking analysis of the core components and key targets. The binding energy between the component and the protein was used to assess the degree of binding. PyMOL software was used to visually display part of the structure.The five components with the highest degrees were identified as core components in the D-C-T-D network, and the 2D structures of the core components were downloaded from PubChem (Paris polyphylla Sm. were mixed at a standard ratio of 5 : 4 and were subjected to reflux extraction twice with 10 times the volume of distilled water for 1 h each. The extracts were then mixed thoroughly and concentrated to a relative density of 1.20–1.25 (70–80°C). The dosage of the C-P drug pair liquid used in subsequent animal studies was 2.7 g/kg/day. Mifepristone was purchased from Beijing Zizhu Pharmaceutical Co., Ltd. . The final dosage was 1.25 mg/kg/day [The herbal medicines were supplied by the Affiliated Hospital of Shandong University of Traditional Chinese Medicine  and verified by Prof. Feng Li. Cinnamomi ramulus and g/kg/day .Rabbit anti-Caspase-3 (bs-0081R) and Caspase-9 (bs-0049R) polyclonal antibodies were obtained from Beijing Bioss Biotechnology Co., Ltd. . A Bax monoclonal antibody (60267-1-Ig) and a Bcl-2 polyclonal antibody (12789-1-AP) were obtained from Wuhan Proteintech Biotechnology Co., Ltd. . PI3 kinase p85 alpha  (ab182651), GAPDH  (ab181602) and Akt  (ab38449) antibodies were obtained from Abcam, Inc. .Forty female Wistar rats and 30 male Wistar rats purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., were housed in the SPF Animal Experimental Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine. All rats had free access to a commercial diet and tap water. All the experimental procedures conformed to the regulations described in the Guide to the Care and Use of Laboratory Animals of the USA National Institutes of Health. The ethical review was approved by the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (AWE-2019-009). According to the random number table method, 40 female Wistar rats were divided as follows: 10 mice served as the blank group, and the remaining 30 were prepared for modeling. Animal models were established according to previous studies . The pitμm-thick sections were prepared for hematoxylin and eosin (H&E) staining. All images were acquired using a NanoZoomer S60 Digital slide scanner .The uterine tissues were fixed in 4% paraformaldehyde solution and embedded in paraffin, and 5 Bax, Bcl-2, Caspase-3, and Caspase-9 protein expressions in uterine tissues were detected using the immunohistochemistry streptavidin-peroxidase (SP) method. The regions were randomly selected under the microscope, and the cumulative optical density, area, and average density of the regions were measured using ImageJ (version 1.8.0).The expression of genes was quantified using the real-time PCR ΔΔCT method with a LightCycler 480II platform . Total RNA was purified using a Simply P Total RNA Extraction Kit according to the manufacturer's instructions. cDNA was synthesized with a ReverTra Ace qPCR RT Kit. Quantitative PCR was performed using a LightCycler 480II Real-Time PCR Detection System. All primers were synthesized by Integrated DNA Technology . The primer sequences for mRNA analysis were as follows: phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA) forward 5′-CTGCAGTTCAACAGCCACAC-3′ and reverse 5′-CCAGCTGCCATCTCAGTTCA-3′; Akt1 forward 5′-AAGGTTTGCTGGGTGAGTGA-3′ and reverse 5′-CTCCTCAGGCGTTTCCACAT-3′. GAPDH expression was used to normalize gene expression.Uterine tissues (50 mg) collected from each group were homogenized and lysed in RIPA lysis buffer and then analyzed to detect the protein concentration using a BCA kit. Tissue total proteins were loaded onto 10% SDS–PAGE gels and transferred onto a PVDF membrane. The membrane was blocked using 5% skim milk and then incubated overnight with primary antibodies  at 4°C. The membrane was then incubated with a secondary antibody. Western blot analysis was performed using a ChemiDoc Imaging System . The density of the signal was quantified by ImageJ.t-tests, whereas the significance of differences among three or more experimental groups was determined by one-way analysis of variance. A value of P < 0.05 was considered to indicate statistical significance. Diagrams were drawn using GraphPad Prism 8.All data were analyzed using GraphPad Prism 8 (version 8.0.2) and are expressed as the mean ± standard deviation (SD). Comparisons between two groups were performed using Student's Paris polyphylla Sm. were obtained from the HERB database and a literature search, and 360 targets corresponding to the active components of Paris polyphylla Sm. were obtained. A total of 384 potential targets of the C-P drug pair were identified after removing the repetition. A total of 504 targets related to AM were obtained through database retrieval. A Venn diagram revealed 45 intersecting targets between the C-P drug pair and AM  and 118 signaling pathways with a GG terms . The resThe 2D structures of the five core components in the D-C-T-D network were obtained from the PubChem database. The 3D structures of 10 key targets in the PPI network were obtained from the RCSB database. Five core components and 10 targets were docked. Binding energy was used to evaluate the degree of binding of ingredients to proteins, and a binding energy <−5 kcal/mol was considered to indicate that component and target could bind. The results of our study showed that the core components had good overall docking with the key targets , indicatIn the control group, the uterine morphology was intact and the muscular layer was continuous. The boundary between the muscular layer and the endometrium was clear, and no gland or endometrium tissue invasion was observed. However, the muscle layer in the model group was thickened with gland invasion and the boundary was blurred. Compared with that in the model group, the muscle layers in the C-P and mifepristone groups were almost intact. The boundary between the muscle layer and the intima layer was fairly clear, and fewer glands invaded the muscle layer (a).The results of real-time PCR analysis indicated that PIK3CA and Akt1 mRNA expression was significantly higher in the AM model group than in the control group. The C-P drug pair and mifepristone significantly suppressed PIK3CA and Akt1 mRNA expression in AM rats compared with that in model rats . The expWe observed changes in the apoptosis-related indicators Bax, Bcl-2, Caspase-3, and Caspase-9 in AM rats after C-P treatment based on tissue- and protein-level detection. The immunohistochemistry results showed that Bax , CaspaseParis polyphylla Sm. are commonly used in TCM [Paris polyphylla Sm. can remove blood stasis. Cinnamomi ramulus and Paris polyphylla Sm. are commonly used to warm meridians, remove blood stasis and relieve pain.Although AM is a condition of benign gynecological lesions, the lesions show abnormal tumor characteristics including endometrial cell proliferation, apoptosis, invasion, and migration. Compared with radical hysterectomy, TCM treatment has positive effects on women's fertility. Cinnamomi ramulus and d in TCM –31. The κB signaling pathway [Paris polyphylla Sm. and its derivatives have been widely used to treat abnormal endometrial bleeding [Paris polyphylla Sm. is also rich in a variety of saponins that have been widely used to combat abnormal cell proliferation and migration and to promote apoptosis of abnormal cells [Modern pharmacological studies have shown that the active components of Cinnamomi ramulus can inhibit the activity, migration, and glycolysis of endometrial stromal cells by inhibiting pyruvate kinase PKM (PKM2) transcription induced by the NF- pathway . Paris pbleeding . Paris pal cells , 34.TCM is characterized by the use of multiple drugs with multiple components and targets for the treatment of diseases. This complexity can hinder the exploration of the molecular mechanisms of TCM drugs in the treatment of AM from the perspective of precision medicine. Network pharmacology is useful for constructing D-C-T-D networks and PPI networks of drug–disease intersection targets based on the abovementioned characteristics of TCM. Through network analysis, the core components and key targets of TCM in the treatment of diseases can be obtained. This research method is beneficial for precision research on TCM and provides an effective means for identifying new targets for disease treatment.In our study, a total of 30 components and 45 corresponding targets of the C-P drug pair related to AM were obtained through network pharmacology. In the D-C-T-C network, ecdysone, pinnatasterone, 20-hydroxyecdysone, flavone and diosgenin exhibited high degree numbers. These results suggest that these compounds may play a major role in the pharmacological mechanism of C-P in the treatment of AM. 20-Hydroxyecdysone has been reported to be a natural active component with the potential to promote apoptosis and autophagy in cancer cells . In addiThrough analysis of the PPI network, we obtained 10 potential core targets of C-P therapy for AM: PIK3CA, IL10, IL6, ESR1, CCL2, TP53, VEGFA, FGF2, PTGS2, and CXCL8. PIK3CA is the catalytic subunit of the dimeric protein PI3K. As the core mediator of the PI3K signaling pathway, PI3K is a critical mediator of cell survival, and its downstream MTOR protein is an important protein involved in autophagy to avoid apoptosis . IL6 andThrough KEGG enrichment analysis of overlapping C-P and AM targets, we found that the PI3K signaling pathway was the most significant nontumor pathway, and GO enrichment analysis showed that phosphorylation (activation) of the PI3K signaling pathway was a significantly enriched biological process. In the molecular docking results, PIK3CA (PI3K) was able to dock with multiple core components. Therefore, PIK3CA may represent an important target of C-P in the treatment of AM. Previous studies have demonstrated that PI3K-Akt activation in AMs promotes endometrial cell proliferation . In addiThe PI3K/Akt signaling pathway is closely related to Bcl-2 family proteins. When stimulated, active Akt leads to the phosphorylation of the proapoptotic protein Bad and inhibits the translocation of Bad from the cytoplasm to the mitochondria, thereby inhibiting mitochondrial dysfunction. Mitochondrial dysfunction stimulates downstream Caspase-3 and Caspase-9. The cleaved forms of Caspase-3 and Caspase-9 regulate other protein substrates, ultimately triggering apoptosis . MoreoveNetwork pharmacology and animal experiments showed that the C-P drug pair can significantly delay disease progression in AM rats. We propose that the pharmacological mechanism of C-P in the treatment of AM is related to inhibition of the PI3K pathway and promotion of apoptosis in AM ectopic lesions. However, unfortunately, the pharmacological components of C-P are not well characterized, and whether C-P regulates apoptosis through the PI3K/Akt pathway remains unclear. We will further explore the effective components and pharmacological mechanism of C-P in the treatment of AM through subsequent experiments.


Myelodysplastic syndrome (MDS) with TP53 mutations has a poor prognosis after transplantation, and novel therapeutic means are urgently needed. Decitabine (Dec) monotherapy has demonstrated improved overall response rates in MDS and acute myeloid leukaemia, although these responses were not durable. This study aimed to preliminary evaluate the efficacy of a Dec-containing allogeneic haematopoietic stem cell transplantation  preconditioning regimen in TP53-mutant MDS. Nine patients with TP53-mutant myelodysplastic syndromes received the decitabine-containing preconditioning regimen and subsequent myeloablative allo-HCT between April 2013 and September 2021 in different centres. At a median follow-up of 42 months , the overall survival (OS) was 89% (8/9), progression-free survival (PFS) was 89% (8/9), and relapse incidence was 11.1%. The incidence of severe acute (grade III-IV) graft-versus-host disease (GVHD) was 22.2% (2/9) and that of chronic moderate-to-severe GVHD was 11.1% (1/9). The 1-year GVHD-free/relapse-free survival (GRFS) was 56% (5/9). In conclusion, we found real-world clinical data that supports the use of a Dec-containing preconditioning regimen before allo-HSCT for possible improved outcomes in TP53-mutant MDS patients; there is therefore an urgent call for an in-depth exploration of the involved mechanism to confirm these preliminary findings. Early studies have shown that 10-day courses of decitabine (Dec) resulted in an increased response rate in MDS with TP53 mutations, but rapid relapse resulted in no long-term benefit for patients . This stNine patients were selected in a consecutive manner between April 2013 and September 2021 in different centres . During this period, study subjects who met the inclusion criteria  were all included. All detected gene mutations were of somatic origin assessed by next generation sequencing (NGS). Detailed data were collected and recorded in standardised electronic forms and integrated by an experienced data administrator.All patients were diagnosed according to the 2016 World Health Organization MDS Classification  and were2/d on day −11 to −9, BU 0.8 mg/kg q6h intravenously on day −7 to −4, and CY 60 mg/kg/day on day −3 to −2. Dec/Flu/Bu was administered as follows: Dec 25 mg/m2/d on day −11 to −9, FLU 30 mg/m2/d on day −8 to −4, and BU 0.8 mg/kg q6h intravenously on day −7 to −4.Myeloablative conditioning regimens were administered to all patients. Dec-containing regimens included Dec plus BU plus CY or Dec plus Flu plus Bu. Dec/BU/CY was administered as follows: Dec 25 mg/m2 on day +1, 10 mg/m2 on day +3, and day +6). Antithymocyte globulin (2.5 mg/kg/day on day -4 to -2), CSA, MMF (1.5 g/m2/day on days 0–28), and a brief course of MTX were administered to patients receiving HLA-mismatched transplants. CSA, a brief course of MTX, MMF, and balliximab (an anti-CD25 antibody) were administered to patients receiving unrelated grafts.Patients who received HLA-matched transplants from siblings were administered cyclosporin A  and methotrexate  was defined as the time from transplantation to the date of relapse or any-cause death. Relapse events were evaluated according to the International Working Group criteria . NRM andThe Kaplan–Meier method was used to calculate survival curves, and the log-rank test was used to compare differences. Normally distributed data are presented as the mean ± standard deviation (SD), whereas non-normally distributed data are presented as the median. Statistical Package for the Social Sciences  for Windows  and GraphPad Prism version 9.2.0 were used for all statistical analyses . A two-tailed p value of < 0.05 indicated statistical significance in all analyses.6/L CD34+ cells. A total of six patients (67%) were diagnosed as MDS-RAEB (refractory anaemia with excess blasts) while the other three patients were diagnosed as MDS-MLD (multiple lineage dysplasia), MDS-RS (ring sideroblast)-MLD and MDS-5q- (MDS associated with isolated del(5q)) respectively. According to the IPSS-R for evaluating prognosis in MDS, four patients (44%) were in poor classification, and five patients (52%) were in very poor. The median patient age at the time of allo-HSCT was 48 years  and the median follow-up time after transplantation was 42 months . At a median follow-up of 42 months , the overall survival (OS) was 88.9% (8/9) , the retTP53 is a key tumour suppressor gene involved in fundamental biological processes required for genomic stability and is recurrently mutated in a subgroup of MDSs and AML . It has The recent findings suggest that the allelic state of TP53 is important for MDS prognosis, and this evaluation can be done by a combination of karyotyping and NGS. There is difference in the outcome when TP53-mutated patients had monoallelic mutations or had multiple hits (multi-hit) consistent with biallelic targeting  and concomitant deletion,mutation(s) and concomitant cnLOH). The evaluation of allelic state TP53 should be done by a combination of karyotyping  and NGS. TP53 multi-hit state predicted risk of death and leukemic transformation independently of the IPSS-R. Even worse, it is more difficult for multi-hit patients to get an effective response after treatments . In thisin vitro suggested that busulfan (BU) plus cyclophosphamide (CY) plus Dec is synergistically cytotoxic to acute myeloid leukaemia (AML) cells regardless of the P53 status (P53-null or P53-wild-type) (The mechanism by which Dec treats TP53-mutant MDS is currently unclear. It has been reasonably suggested that Dec may increase the sensitivity of AML/MDS patients with TP53 mutations to treatment . Howeverld-type) . They exld-type)  and enhald-type) , 38–40 oTo the best of our knowledge, there has no published data of azacytidine, another hypomethylating agent, as part of the preconditioning regimen before HSCT. Considering the extremely poor prognosis of patients with TP53 mutations, this paradigm of combination therapy also provides a novel strategy to reinforce the efficacy for other new drugs, such as MDM2 inhibitors, magrolimab, and sabatolimab. However, its rationality as part of a preconditioning regimen should be validated in preclinical studies first.Although our report shows that there might be a subset of patients with TP53-mutant MDS, who derive benefit from Dec-containing conditioning regimen transplantation, it has limitations owing to its limited sample size. For example, the high rate of severe (grade III-IV) acute GVHD in our cohort  may also be caused by its statistical limitation of small sample size. However, given the low incidence of TP53 mutations in MDS, the high median age at onset , and the significantly increased detection rate of TP53 mutations in elderly patients , greaterIn conclusion, our real-world clinical data support that Dec-containing preconditioning regimen before allo-HSCT is highly effective in TP53-mutant MDS patients and urgently call for mechanism and randomised prospective clinical studies to confirm these preliminary findings.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by the ethics committee of the Fifth Medical Centre of the PLA General Hospital. The patients/participants provided their written informed consent to participate in this study.YW and YS performed the research and drafted the manuscript. JX was involved in the selection and management of the patient and in the manuscript review. LH and YL were involved in the identification, selection, and management of the patient and manuscript review. YT and LD provided Clinical samples of other centres. LQ and BZ put forward the modification suggestion. JH, NL, JC, BL, SL, JN, LW, ZQ, YZ, and JR collected and analysed the data. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Program of Army Logistics Research (AWS17J010) and the National Natural Science Foundation of China (82100239).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Correspondingly, the growth of Pt particles is reduced from 0.95 nm  to 0.08 nm; that is, the coalescence of Pt particles is effectively alleviated upon using graphitized carbon black.The anti-corrosion properties of the carbon substrates of cathode catalysts play a vital role in the commercialization of fuel cell vehicles. Our report reveals the enhanced durability of graphitized carbon black catalyst substrates in polymer electrolyte membrane fuel cells (PEMFCs), tested under simulated start-stop cycling and high potential holding conditions. Graphitized carbon treated at various temperatures is used as the support for Pt catalysts. The catalyst utilizing graphitized carbon treated at 1800 °C demonstrates superior antioxidation properties and the inhibition of Pt particle coarsening. The decay ratio of the potential at 1000 mA cm The enhanced anti-corrosion properties of graphitized carbon substrates are evaluated via accelerated stress tests (ASTs). The graphitized surface properties of carbon are responsible for alleviating the coarsening and agglomeration of Pt particles. The platinum particles will migrate and agglomerate, finally detaching from the carbon support.20,21 The interfacial structure formed between the Pt nanoparticles and carbon support changes because of the functional groups generated on the carbon surface.22,23 The electrons in Pt nanoparticles near the oxygen functional groups can be transferred to the electronegative oxygen atoms, increasing the oxidation state of Pt; this can trigger the easy dissolution of Pt nanoparticles and accelerate the Ostwald ripening process.24,25 Meanwhile, the conductivity of the carbon support decreases; as a result, the impedance of the fuel cell increases.20 In addition, the carbon support will become hydrophilic, which can have an impact on gas permeability as well as water management. Therefore, high constant potential or potential cycling tests are usually conducted as protocols to investigate the effects of carbon support corrosion on PEMFC performance. Protocols proposed by the US Fuel Cell Council (USFCC),19 US Department of Energy (DOE),26 and the Fuel Cell Commercialization Conference of Japan (FCCJ)27–29 are widely used as standard protocols. Potential undulation during the start-stop protocol can be simulated via potential cycling to accelerate the degradation of the catalyst support within a short period of time.30,31For automobile applications, the local cathode potential can reach as high as 1.5 V during starting and stopping due to the H32,33 Jung et al. employed graphitized carbon black and a N-doped graphitic carbon matrix as supports to increase corrosion resistance and to introduce N-modified active sites onto the carbon surface.34,35 Chen developed nitrogen/metal co-doped graphene tubes as substrates for stable ORR catalysts.36 Highly graphitized carbon materials, such as carbon nanofibers and carbon nanotubes, have been employed due to their unique corrosion resistance properties. However, the chemically inert planes of carbon nanotubes lead to low Pt nanoparticle dispersibility, large crystallite/particle sizes, and ease of agglomeration, resulting in the rapid and massive loss of the electrochemically active surface area (ECSA).37,38 It has been observed that the agglomeration of Pt nanoparticles mainly occurs at the junctions and edges of graphited carbon particles, based on electron tomography.39 Therefore, determining the degree of graphitization of the carbon support appears to require an optimum balance between uniform metal-particle dispersion and sufficient carbon corrosion resistance in order to meet the requirements of the various operating conditions of automotive PEMFCs, especially operation conditions that cause high potentials.To address the above-mentioned stability issues affecting carbon carriers, a lot of effort has been made to improve the inherent properties of ORR catalysts; in these studies, substitution and surface modification and coating are mainly employed.via transmission electron microscopy (TEM).In this work, the degree of graphitization of the carbon support was adjusted, and the resulting materials were used to synthesize platinum-based catalysts. The durability of single cells with the prepared catalysts located at the cathode was examined using accelerated stress test (AST) protocols, including under potential-cycling simulated start-stop and high potential holding conditions. The structural changes in the carbon supports and Pt nanoparticles were monitored 2.2.1.via heat treatment under N2 protection . KetjenBlack EC-600JD was thoroughly premixed with Ni(NO3)2  before heat treatment to promote the graphitization reaction of carbon black and reduce the temperature required for graphitization. The details are as follows. Appropriate amounts of EC-600JD and Ni(NO3)2, based on a C : Ni mass ratio of 20 : 1, were evenly dispersed in ultrapure water (18.2 MΩ cm at 25 °C) via ultrasonic vibration. The well-mixed suspension was transferred to a rotary evaporator  and subjected to treatment at 100 °C to obtain dried powder. The powder was then heat-treated for 3 h at a predetermined temperature (1600 °C or 1800 °C) at a heating rate of 20 °C min−1 in a vacuum furnace  under a N2 atmosphere. Finally, the heat-treated powder was washed, filtered, dried, and labeled as EC-G1600 or EC-G1800, depending on the treatment temperature.The degree of graphitization of carbon black  was increased 2.2.i.e., Pt/EC-x (where x is G1600 or G1800), were synthesized via the one-pot ethylene glycol  reduction of the Pt precursor H2PtCl6·6H2O . The pH value of the ethylene glycol solution was adjusted to 12 with NaOH  before being heated to 130 °C under a N2 atmosphere and maintained for 3 h. After cooling to room temperature, the obtained solid was filtered with abundant ultrapure water and dried in a vacuum oven for 12 h. For comparison, Pt supported on raw carbon black (EC-600JD) was prepared using the same method and labelled as Pt/EC. Commercial Pt/C  was used as received and labelled as Pt/C-JM.The catalysts supported on the as-prepared graphitized carbon black samples, 2.3.TEM analysis  was conducted at 200 kV to observe the fresh Pt/C catalysts and aged Pt/C catalysts that underwent durability testing. Raman spectroscopy  analysis was carried out with radiation from an argon laser (514 nm) to examine the structures of the raw and graphitized carbon black samples. Specific surface area and porosity measurements using all carbon black samples were performed with a volumetric device . X-ray diffraction  was used with Cu Kα radiation to examine the as-prepared Pt-based catalysts.2.4.2) coated with the corresponding catalyst was used as the working electrode. A reversible hydrogen electrode (RHE) and Pt wire were used as the reference and counter electrodes, respectively.Electrochemical tests were carried out with a CHI660D potentiostat (CH Instruments). A glassy carbon rotating disk , where Q is the integral charge of the H desorption peak after removing the baseline of the double-layer area, m is the mass of Pt loaded on the working electrode, and k is equal to a constant of 0.21 mC cm−2, which represents the average amount of charge for removing a single layer of H atoms from a pure Pt surface. The current was normalized to the Pt amount to get the mass activity.Homogeneous catalyst ink was prepared as follows. 2 mg of the studied catalyst was dispersed in 1 mL of a mixed solvent containing isopropanol  and an ionomer  (the mass ratio was 30 : 1), and this was sonicated for 30 min. 10 μL of ink was transferred onto the rotating disk. The cyclic voltammetry (CV) technique was used to determine the electrochemically active surface area (ECSA) 2.5.via spraying catalyst ink onto Nafion® 212 membranes (DuPont) with the heating platform set at 80 °C, and then one piece of gas diffusion layer  was physically placed on each side of the catalyst-coated membrane. The Pt loading of the cathode catalyst layer is 0.4 mgPt cm−2. The cathode catalyst ink was obtained via dispersing the studied catalyst into a mixed solvent made up of ultrapure water, isopropanol, and an ionomer dispersion , and this was sonicated for 30 min. The ratio of catalyst to Nafion® ionomer was 3 : 1. All anode catalyst layers were prepared using commercial Pt/C-JM with the same method as the cathode and with Pt loading of 0.2 mgPt cm−2. The active electrode area of the obtained single cell is 25 cm2.Single-cell tests were conducted using a fuel cell test bench . Membrane electrode assemblies (MEAs) were prepared During the polarization curve testing of single cells, pure hydrogen and compressed air were supplied to the anode and cathode sides with stoichiometric ratios of 1.5 (anode) and 3.8 (cathode); a backpressure of 100 kPa and relative humidity (RH) of 80% were employed at both sides. The temperature of the cell was maintained at 80 °C using a water thermostat.2.6.via employing AST protocols. For electrochemical environment testing, triangle wave potential cycling between 1 and 1.5 V vs. RHE with a sweep rate of 500 mV s−1 was employed to simulate the high potential conditions a fuel cell stack may encounter.40,41 For single-cell testing, a square wave between 0.9 V and 1.3 V (vs. anode) with a frequency of 1 min per cycle was used to simulate the conditions experienced by a MEA during start-stop operations.30,31 The AST protocols are shown in via holding the cathode potential at 1.4 V for 30 min.42The carbon-corrosion effects and degradation behaviors of the catalysts were scrutinized 3.3.1.3.1.1.θ values of 25° and 43° are assigned to the (002) and (101) diffraction planes of graphitic carbon, respectively.43 Compared to the clear patterns of the graphitized samples (EC-G1600 and EC-G1800), the signals of the raw material (EC-600JD) are immensely broadened, revealing the presence of large amounts of amorphous carbon in the material.44 The graphitic reflections become more legible after graphitizing, and they become even more well-resolved as the graphitization temperature rises. Raman spectroscopy is an available analytical tool for researching the inherent properties of carbonaceous materials. As shown in −1) and a G (∼1580 cm−1) peak. The D-to-G peak intensity ratio (ID/IG) is merely 1.09 for EC-600JD, but it is effectively strengthened to 1.28 for EC-G1600 and further to 1.37 for EC-G1800. The value of ID/IG displays an overall tendency to increase from the raw material EC-600JD to the final two graphitized carbon products. According to the literature,45 the ID/IG ratio from the Raman spectrum depends on the amorphization trajectory, ranging from perfect graphite sheets to highly amorphous sp3 carbon; this consists of three stages: (1) from graphite to nanocrystalline graphite; (2) from nanocrystalline graphite to a-C; and (3) from a-C to ta-C.The crystallinities and structures of the EC-600JD raw material and graphitized products were first inspected using XRD, as shown in ID/IG increases according to the TK equation:46where C (515.5 nm) is ∼44 Å and La is the cluster diameter or in-plane correlation length. In stage (2), defects are gradually introduced into the graphite layer. The end of this stage corresponds to complete disorder. ID/IG decreases with increasing amorphization. In stage (3), ID/IG is very low or 0. During the graphitization process, some of the amorphous state progressively transforms into nanocrystalline graphite. Defects inevitably exist in the graphite layer of the final graphitized carbon black product. Therefore, the amorphization trajectory for all samples are in stage (2). ID/IG will increase with decreasing disorder. Thus, a higher ID/IG ratio for graphitized carbon means less disorder in the graphitic bonds.In stage (1), 3.1.2.47 The response in the relative pressure (P/P0) range from 0 to 0.98 consists of three stages: low-pressure (0–0.03); medium-pressure (0.03–0.86); and high-pressure (0.86–0.98) areas. The micropores are mainly filled in the low-pressure area. Compared with untreated EC-600JD, the slopes of the absorption–desorption curves of EC-G1600 and EC-G1800 in this region decrease, which indicates that the microporous structure of the graphitized carbon is reduced significantly. In the medium-pressure region, the hysteresis loops caused by the capillary condensation of nitrogen molecules reveal the mesoporous structures of all the samples.48 In the high-pressure region, the hysteresis loops of the three types of carbon support reveal the existence of mesoporous structures. All of the carbon supports are dominated by mesoporous structures. The Brunauer–Emmett–Teller (BET) surface areas of the three samples were calculated to be 1360 m2 g−1, 634 m2 g−1, and 458 m2 g−1 for EC-600JD, EC-G1600, and EC-G1800, respectively. The pore volume distributions were evaluated via the Barrett–Joyner–Halenda (BJH) method, and they are provided in 3.1.3.x catalysts with the representative diffraction peaks. The corresponding crystal planes are marked in 3.2.2-saturated 0.1 M HClO4 before and after 50 000 cycles are shown in vs. RHE) in the CV curves. The ECSAs of each sample before and after ASTs were calculated using the process outlined in the Experimental section, and the results are summarized in 25 The corresponding ECSA decay levels, normalized to the initial ECSAs, of the catalysts as a function of the number of cycles are given in CV scans from the as-prepared catalysts obtained in N3.3.3.3.1.To simulate the operating environment of the start-stop conditions that PEMFCs are constantly subjected to during the actual operation of fuel cell vehicles, we used start-stop cycles to evaluate the durability of the cathode catalysts in PEMFCs. The initial polarization curves of single cells prepared with the four catalysts as cathode catalysts are shown in 3.3.2.0 phase content because of the existence of fewer oxygen functional groups on the carbon surface, resulting in more electrochemically stable Pt nanoparticles. On the surface of untreated carbon black, the electrons of the Pt nanoparticles near the oxygen functional groups migrate toward the negative oxygen atoms, leading to an increased level of Pt nanoparticles in an oxidized state, triggering ready dissolution and Ostwald ripening.25 It can be observed from the high-resolution TEM images of the Pt/EC-G1600 and Pt/EC-G1800 catalysts before and after start-stop ASTs in 49 After start-stop ASTs, the graphite layer structure of the Pt/EC-G1600 catalyst carrier had mostly disappeared. The Pt/EC-G1800 carbon catalyst carrier retained its intact layer structure. The results indicate that the carbon support of the Pt/EC-G1600 catalyst begins to undergo gradual oxidation during the start-stop protocol, resulting in the gradual destruction of the graphite lattice structure. The carbon carrier of Pt/EC-G1800 has a complete graphite lattice structure, with the oxidation resistance being improved. It is indicated that the performance degradation of the cathode catalyst mainly results from the coarsening of the platinum particles, which is caused by the corrosion of the carbon support during start-stop cycling. After the graphitization of the carbon support, the growth of the platinum particles is effectively suppressed. Generally, the carbon supports of cathode catalysts are extremely corroded as a result of start-stop cycling, causing the pronounced detachment and aggregation of the Pt nanocrystallites.50 For Pt/C-JM, start-stop cycling resulted in the preferential oxidation of the chaotic domains of the carbon support. However, in the case of the graphitized carbon supports, the graphite lattice inhibits the oxidation process.TEM images of the catalysts before and after the start-stop AST protocol are exhibited in −2 and the decay rates are shown in The high potential is at the root of the oxidation of carbon carriers. High potential operation tests were further conducted to evaluate the durability of the single cells. The polarization curves of the single cells before and after high-potential ASTs are provided in 51 During the high potential stress test protocol, the degradation of commercial Pt/C-JM with an amorphous carbon support structure was observed to be more rapid. Compared to the Pt/EC-G1600 catalyst, the carbon support of the Pt/EC-G1800 catalyst has a finer graphite lattice, so the kinetic oxidation rate is the lowest.Similar to the morphologies of the catalysts during start-stop ASTs, the platinum particles of Pt/C-JM were severely coarsened, but the platinum particles supported on graphitized carbon did not significantly grow or coarsen after the high-potential AST protocol, as shown in 4.The degradation of as-prepared catalysts with graphitized carbon supports was investigated using both simulated start-stop cycling and high-potential AST protocols. The Pt/EC-G1800 catalyst with the carbon support with the most complete graphite lattice was more resistant to electrochemical corrosion than catalysts with a defective graphite lattice support (Pt/EC-G1600) or structurally disordered carbon supports (Pt/EC and Pt/C-JM) during both stress testing protocols.vs. 38.6%). According to the results following start-stop and high-potential AST protocols using single cells, the catalyst particles on the graphitized carbon supports underwent the smallest amounts of growth among all the samples. Meanwhile, the Pt/EC-G1800 catalyst maintained the maximum performance, with a decay rate of 0.50% compared to 34.9% for the Pt/C-JM catalyst, during high-potential ASTs. Moreover, the graphite lattice structure of its carbon support remained intact. During start-stop ASTs, the Pt particles of the Pt/EC-G1800 catalyst grew by 0.66 nm compared to 1.57 nm for commercial Pt/C-JM.During RDE testing, the Pt/EC-G1800 catalyst displayed the lowest amount of ECSA loss and the lowest attenuation rate, less than half of the decay rate of commercial Pt/C-JM (18.1% Therefore, using a graphitized carbon support can effectively improve the antioxidation capabilities, alleviating the coarsening and agglomeration of Pt nanoparticles. Graphitized carbon could act as a more stable catalyst support and offer an alternative solution under the demanding operating conditions faced by automotive PEMFCs.No potential competing interests are reported by the authors.